During FY2013: 1) We adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrPSc (PrPRes). Scrapie brain dilutions up to 10-8 and 10-13 were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrPRes levels, and found that the former were similar. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrPRes levels. We also found that eQuIC, which incorporates a PrPSc immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrPSc. 2) We adapted the RT-QuIC assay to the detection of prion seeding activity in the saliva of deer infected with chronic wasting disease. 3) We have also collaborated with neurologists to begin evaluating the utility of our previously developed RT-QuIC assay for the detection and diagnosis of human sporadic Creutzfeldt-Jakob disease using specimens derived from blood or olfactory mucosa brushings. The data so far have shown that RT-QuIC sensitively detects prion seeding activity in nasal brushings from a wide variety of human CJD cases. Results from the blood testing are pending. We are continuing to test additional samples that are being collected by our collaborators. Conclusions as to the diagnostic utility of these tests must await further testing.